The proteasome is a large, ATP-dependent, multi-subunit, barrel-shaped N-terminal nucleophile hydrolase present in the cytosol and nucleus of eukaryotic cells, and is responsible for the degradation of the majority of cellular proteins (Baumeister et al., “The Proteasome: Paradigm of a Self-Compartmentalizing Protease,” Cell 92:367-380 (1998); Goldberg, A. L., “Functions of the Proteasome: From Protein Degradation and Immune Surveillance to Cancer Therapy,” Biochemical Society Transactions 35:12-17 (2007)). The proteasome not only controls many critical cellular checkpoints through degradation, but also generates peptides for antigen presentation (Goldberg, A. L., “Functions of the Proteasome: From Protein Degradation and Immune Surveillance to Cancer Therapy,” Biochemical Society Transactions 35:12-17 (2007); Rock et al., “Inhibitors of the Proteasome Block the Degradation of Most Cell Proteins and the Generation of Peptides Presented on MHC Class I Molecules,” Cell 78:761-771 (1994)). Highly specific proteasome inhibitors can markedly limit the overall supply of peptides for MHC class I molecules and thus block antigen presentation (Rock et al., “Protein Degradation and the Generation of MHC Class I-Presented Peptides,” Advances in Immunology 80:1-70 (2002)). The constitutive proteasome core particle is called 20S (c-20S) because of its sedimentation properties. Inside the c-20S core reside two copies of each of three proteases with distinct specificities, β1 (caspase-like), β2 (tryptic-like) and β5 (chymotryptic-like) (Bedford et al., “Ubiquitin-Like Protein Conjugation and the Ubiquitin-Proteasome System as Drug Targets,” Nature Reviews. Drug Discovery 10:29-46 (2011)). However, lymphocytes and cells that have responded to interferon-γ express a different proteasome, called the immunoproteasome (i-20S), in which the corresponding proteases are the products of different genes: β1i, β2i and β5i. Intermediate proteasomes that contain mixed β subunits are found in many cells, for example in the mucosa of the colon and small bowel (Guillaume et al., “Two Abundant Proteasome Subtypes that Uniquely Process Some Antigens Presented by HLA Class I Molecules,” Proc. Nat'l Acad. Sci. USA 107:18599-18604 (2010)). The effects of replacement of constitutive subunits by immuno-β subunits include increased proteolytic activity and altered peptide preferences of the active sites (Rock et al., “Proteases in MHC Class I Presentation and Cross-Presentation,” Journal of Immunology 184:9-15d (2010)). For example, the caspase-like β1 replacement, β1i, preferentially cleaves after small hydrophobic residues rather than after aspartate (Huber et al., “Immuno- and Constitutive Proteasome Crystal Structures Reveal Differences in Substrate and Inhibitor Specificity,” Cell 148:727-738 (2012)). This results in altered peptide products, such that mice with combined deficiency of β1i, β2i, and β5i are viable, fertile and healthy but express a different antigenic peptide repertoire than wild type mice, as evidenced by their rejection of syngeneic wild type splenocytes (Kincaid et al., “Mice Completely Lacking Immunoproteasomes Show Major Changes in Antigen Presentation,” Nature Immunology 13:129-135 (2012)). Hu c-20S and i-20S appear to regulate cytokine production through different pathways (Muchamuel et al., “A Selective Inhibitor of the Immunoproteasome Subunit LMP7 Blocks Cytokine Production and Attenuates Progression of Experimental Arthritis,” Nature Medicine 15:781-787 (2009)). Hu c-20S controls the activation of NF-κB via the degradation of IκB, the binding partner of NF-κB in the cytosol (Perkins, N.D., “Integrating Cell-Signalling Pathways with NF-[Kappa]B and IKK Function,” Nat. Rev. Mol. Cell Biol. 8:49-62 (2007)), and inhibition of c-20S blocks the activation of NF-κB (Meng et al., “Epoxomicin, a Potent and Selective Proteasome Inhibitor, Exhibits In Vivo Antiinflammatory Activity,” Proc. Nat'l Acad. Sci. USA 96:10403-10408 (1999)). For its part, among other potential pathways, i-20S appears to control the co-translocation of TLR9 and Unc93B1, an endoplasmic reticulum (ER)-resident protein, to endosomes (Hirai et al., “Bortezomib Suppresses Function and Survival of Plasmacytoid Dendritic Cells by Targeting Intracellular Trafficking of Toll-Like Receptors and Endoplasmic Reticulum Homeostasis,” Blood 117:500-509 (2011)). Proteasomes control diverse cellular functions, among them signal transduction for inflammatory cytokine release, antigen presentation, and the ability of plasma cells to secrete antibodies without dying from accumulation of misfolded immunoglobulins (Goldberg, A. L., “Functions of the Proteasome: From Protein Degradation and Immune Surveillance to Cancer Therapy,” Biochemical Society Transactions 35:12-17 (2007); Bedford et al., “Ubiquitin-Like Protein Conjugation and the Ubiquitin-Proteasome System as Drug Targets,” Nature Reviews. Drug Discovery 10:29-46 (2011); Neubert et al., “The Proteasome Inhibitor Bortezomib Depletes Plasma Cells and Protects Mice with Lupus-Like Disease from Nephritis,” Nature Medicine 14:748-755 (2008)). Thus the proteasome could be a target for treating autoimmune and inflammatory diseases. For example, inhibition of the proteasome in plasmacytoid dendritic cells (pDCs) prevents the trafficking of TLRs, resulting in a block of nuclear translocation of IRF-7, consequently suppressing the production of IFNα (Hirai et al., “Bortezomib Suppresses Function and Survival of Plasmacytoid Dendritic Cells by Targeting Intracellular Trafficking of Toll-Like Receptors and Endoplasmic Reticulum Homeostasis,” Blood 117:500-509 (2011)), a cytokine implicated in systemic lupus erythematosus (SLE). However, by the same token, widespread inhibition of proteasomes can be expected to be toxic and has proven toxic in the clinic.
Two proteasome inhibitors approved by the FDA for treatment of malignancy, Bortezomib and Carfilzomib, inhibit both the c-20S β5c and the i-20S β5i (Huber et al., “Inhibitors for the Immuno- and Constitutive Proteasome: Current and Future Trends in Drug Development,” Angewandte Chemie 51:8708-8720 (2012)). Bortezomib, a dipeptidyl boronate, is a slow-binding, covalent but reversible inhibitor, whereas Carfilzomib is a peptide with an epoxyketone warhead that inhibits proteasomes irreversibly. In addition to treatment of malignancy, Bortezomib has been reported to be effective in inflammatory bowel disease (IBD), SLE, graft-versus-host disease, antibody-mediated graft rejection, rheumatoid arthritis (RA), and other immunologic, autoimmune and/or inflammatory conditions. However, such a broad-spectrum inhibitor is too toxic for chronic treatment of non-malignant diseases. ONX 0914, another peptide epoxyketone, has modest selectivity for i-20S β5i (Muchamuel et al., “A Selective Inhibitor of the Immunoproteasome Subunit LMP7 Blocks Cytokine Production and Attenuates Progression of Experimental Arthritis,” Nature Medicine 15:781-787 (2009)) and is reported to have efficacy in rheumatoid arthritis (Muchamuel et al., “A Selective Inhibitor of the Immunoproteasome Subunit LMP7 Blocks Cytokine Production and Attenuates Progression of Experimental Arthritis,” Nature Medicine 15:781-787 (2009)), SLE (Ichikawa et al., “Beneficial Effect of Novel Proteasome Inhibitors in Murine Lupus Via Dual Inhibition of Type I Interferon and Autoantibody-Secreting Cells,” Arthritis and Rheumatism 64:493-503 (2012)), experimental colitis (Basler et al., “Prevention of Experimental Colitis by a Selective Inhibitor of the Immunoproteasome,” Journal of Immunology 185:634-641 (2010)), and multiple sclerosis (Basler et al., “Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis,” EMBO Mol. Med. 6:226-238 (2014)). Nonetheless, it, too, acts irreversibly and has considerable toxicity.
The present invention is directed to overcoming these and other deficiencies in the art.